Transport mechanism for succinate and phosphate localized in the plasma membrane of bovine spermatozoa

Abstract

Bovine spermatozoa accumulated a small amount of 32Pi during aerobic incubation in vitro. At least 50% of the acquired isotope rapidly entered cellular nucleotides. Both adenosine and guanosine di- and triphosphates were labeled, but contrary to expectations, the specific activity of ADP exceeded that of ATP. The uptake of phosphate and its incorporation into nucleotides were suppressed by respiratory inhibitors and were abolished by treatment with sulfhydryl-directed reagents at 10 to 20 nmol/mg of sperm protein. With fructose as an energy source for motility, glycolysis did not support phosphate uptake. Nucleotide labeling was increased 60 to 80-fold when the cells were treated with the polyene antibiotic filipin, and filipin was able to reverse the inhibition of phosphate (and succinate) entry produced by N-ethylmaleimide or mersalyl. Since filipin interacts specifically with the cholesterol-containing plasma membrane of bovine spermatozoa and increases its permeability, it is probable that the plasma membrane normally limits phosphate and succinate transport into these cells. This contention is further supported by the observation that high concentrations of extracellular Pi, the penetration of which was extremely limited under these conditions, protected against inactivation by N-ethylmaleimide. Phosphate uptake was increased 10 to 20-fold, but nucleotide labeling was inhibited, when calcium was present in the incubation medium. Ruthenium red, presumably acting extracellularly, prevented these effects of calcium. Thus, the entry of phosphate and succinate into spermatozoa is controlled by plasma membrane components that resemble the phosphate and succinate exchangers and calcium carrier found in mitochondria isolated from other sources.